General Considerations
The general purpose of chromatographic and ion exchange separations is to isolate a molecule or ion from solution and from other species. Chromatographic and ion exchange separations occur by contacting a solution with a solid support that has a surface with defined chemical characteristics. The separation occurs by partitioning of the solutes between the solution and the solid phase, which occurs because the surface of the support interacts selectively with a desired solute molecule or class of solute molecules, and the desired solutes are adsorbed. Passage of a solution through a bed or column of the solid support results in retardation of the adsorbed molecules and, thereby, separation of the desired compounds from others. The effectiveness and selectivity of the separation process is a function of several factors. These factors include the relative affinities of the solutes for the surface and the extent to which equilibration of a solute between solution and the solid phase reaches equilibrium.
The technology and science of chromatographic supports is imperfect, and existing products are a result of trade offs of a support design parameters. The capacity of a solid support is a function of the surface area of the support particles. The surface area of porous particles is inversely related to the pore size of the particles. Small pore diameters relate to increased surface area, and the number of functional groups available to interact with the desired solute molecules. This is referred to as capacity. Unfortunately, porous particles with small pore sizes and large surface areas are not very permeable to the flow of solutes in and out of the pores, where the adsorption occurs. Recognition of this fact has stimulated development of particles with larger pore diameters that are more permeable to solution flow. More importantly, the larger pores permitted the chromatography of macromolecules, such a proteins. The tradeoff suffered with the increased pore diameters is reduced surface area, and proportionately lower capacity. In addition to the pore size, surface area, and capacity issues that require compromises in support design, all conventional chromatographic supports and catalysts suffer from sluggish adsorption-desorption kinetics that result from slow diffusion of solute molecules through the stagnant boundary layer at the solid-liquid interface.
Previous Efforts of Solid Support Improvement
Girot and Boschetti (U.S. Pat. No. 5,559,453) disclose modified porous supports for chromatography biomolecules. The support is prepared by use of a passivation mixture, comprising a main monomer, a passivating monomer, and a cross linking agent, which mixture upon polymerization results in the substantial elimination of the undesirable nonspecific interaction with biomolecules. The matrix is prepared by first adsorption of various bifunctional compounds to the support surface. The bifunctional molecules, called the passivating monomers, adsorb by virtue of charge interactions and/or hydrogen binding to the silanol surface of the porous support. The passivating monomers include diethylaminoethyl methacrylamide and methacrylamidopropyl trimethyl ammonium chloride, which are cationic at pH>7.0, and will form ion pairs with the deprotonated silanol surface. The result of adsorbing the passivating monomer, is that the surface becomes coated with the passivating monomer and the copolymerizable vinyl group of the molecule is oriented toward the solution in contact with the surface. The polymerization mixture, containing a functional monomer, a crosslinking agent, an initiator, and a porogen is then permitted to polymerize in the pore of the support to form a highly crosslinked gel structure, or the so-called gel in a shell. It is probable that the passivating monomer copolymerizes with the other monomers provided, resulting covalent bonds between the passivating layer and the support. The porogen is necessary to provide pores or channels for solution and analyte molecules to flow through the gel at an acceptable pressure differential and velocity. After completion of the polymerization, the support is washed to remove unreacted monomers and porogen.
Although the passivated porous supports have good properties and have achieved commercial acceptance, there are some underlying design flaws in these materials that limit their performance. The first problem is the noncovalent association of the passivating monomer with the porous support. It is highly unlikely that all of the passivating monomers are incorporated in the polymer formed inside the pores. The result of this is that the passivating monomer is subject to leaching under changes in mobile phase pH changes. Leaching of the passivating surface coating can lead to patches of silanol surface that have no protective coating. This in turn will cause nonspecific binding with proteins that are well know to interact with silanol surfaces. The second design flaw of the passivated porous supports is the crosslinking density of the gel inside the pores. The highly crosslinked copolymer does not permit facile flow of solution through the pores, and it is necessary to use porogens to create micro channels that permit fluid exchange to occur. Such a gel structure will not have the optimal flow characteristics and exchange kinetics.